Gastroenterology

Gastroenterology

Volume 117, Issue 4, October 1999, Pages 770-775
Gastroenterology

Rapid Communications
Molecular characterization and functional regulation of a novel rat liver-specific organic anion transporter rlst-1☆,☆☆,★

https://doi.org/10.1016/S0016-5085(99)70333-1Get rights and content

Abstract

Background & Aims: Recently, we isolated a new complementary DNA (cDNA) encoding human liver-specific organic anion transporter (LST-1), representing the multispecificity of human liver. The aim of this study was to isolate a rat counterpart of human LST-1 and examine the expression regulation of its messenger RNA (mRNA) to clarify the molecular basis of cholestasis. Methods: A rat liver cDNA library was screened with human LST-1 cDNA as a probe. Xenopus oocyte expression system was used for functional analysis. Northern blot analyses were performed using the isolated cDNA (termed rlst-1). The bile duct ligation model and the cecum ligation and puncture model were used for expression analyses. Results: rlst-1 encodes 652 amino acids, predicting at least 11 transmembrane regions. The overall homology with human LST-1 was 60.2%, which is the highest among all known organic anion transporters. rlst-1 also belongs to the same new gene family as human LST-1, located between the organic anion transporter family and the prostaglandin transporter. rlst-1 preferably transports taurocholate (Km, 9.45 μmol/L) in an Na+-independent manner. The rlst-1 mRNA is exclusively expressed in the liver. In both the bile duct ligation model and the cecum ligation and puncture model, mRNA expression levels of rlst-1 were down-regulated. Conclusions: rlst-1 is a counterpart of human LST-1 and is one of the important transporters in rat liver for the clearance of bile acid. The expression of rlst-1 may be under feedback regulation of cholestasis by biliary obstruction and/or sepsis.

GASTROENTEROLOGY 1999;117:770-775

Section snippets

Isolation of rat rlst-1 complementary DNA

A rat liver complementary DNA (cDNA) library was constructed, and 2.5 × 105 independent clones were hybridized with the full length of human LST-1 in a formamide (25%) solution at 42°C as described previously.14 Filters were washed in 2× standard saline citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) at 50°C for 1 hour. Six positive clones were isolated and rescued into pBluescript SK(−). The cDNA inserts of these clones showed an identical restriction enzyme digestion pattern except for

Isolation and structural analysis

The isolated cDNA encoding rat rlst-1 consisted of 652 amino acids (Mr, 72,762) (Figure 1A).

. (A) Alignment of deduced amino acid sequence of rat rlst-1 and human LST-1. The sequences are aligned with single-letter notation by inserting gaps (−) to achieve the maximum homology. The putative TM segments were assigned. Sequence motifs for potential N-glycosylation sites (triangles) and possible phosphorylation sites (*) are indicated. (B) Hydrophobicity profile was drawn using the algorithm of

Acknowledgements

The authors Dr. Kazuo Nunoki for discussions and Emiko Shibuya for technical assistance.

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    ☆

    Address requests for reprints to: Takaaki Abe, M.D., Department of Neurophysiology, Tohoku University School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan. e-mail: [email protected]; fax: (81) 22-717-8154.

    ☆☆

    Supported in part by research grants from the Ministry of Education, Science, and Culture of Japan; Yamanouchi Foundation for Research on Metabolic Disorders; Nishimiya Foundation; Tokyo Biochemical Research Foundation; Japan Research Foundation for Clinical Pharmacology; Yokoyama Foundation for Clinical Pharmacology; Japan Health Sciences Foundation; and Inamori Foundation.

    ★

    The sequence of rat lst-1 has been deposited under the GenBank accession number AF147740.

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    Copyright © 1999 American Gastroenterological Association. Published by Elsevier Inc. All rights reserved.